The goal of the proposed research is to isolate and analyze the functions of proteins that interact with the following sequence elements of eukaryote chromosomes: 1. Upstream Activating Sequences (UAS's) GAL 4 and GAL 80 proteins, which mediate induction and repression of transcription by the GAL family of UAS's in yeast, will be purified and characterized. These proteins have been detected in crude extracts with a nitrocellulose filter-binding assay, and GAL 4 protein has been purified 220-fold on the basis of this assay. The postulated interaction of the proteins will be investigated with purified preparations and an effort will be made to reconstitute the regulation of transcription in vitro. 2. Enhancers Preliminary findings indicative of gene activation by an immunoglobulin enhancer in yeast will be pursued with additional enhancers and enhancer mutants. An effort will be made to detect enhancer-binding proteins in yeast and identify their homologues in mammalian cells. Preliminary evidence for enhancement of transcription by a UAS in mammalian cells will be pursued with additional UAS's, fragments of UAS's, and flanking sequences. 3. Centromere (CEN) Sequences A protein that binds to centromere DNA element I (CDE I) of yeast will be purified and characterized. The functional significance of the binding may be revealed by genetic manipulations of CDE I at a site adjacent to a UAS. Any relationship to a centromere antigen reactive with sera of scleroderma patients will be investigated. Similar studies will be undertaken of CDE II and CDE III-binding proteins. 4. Autonomously Replicating Sequences (ARS's) A binding activity specific for domain B of ARS 1 will purified and characterized. Activities specific for domains A and C will be investigated as well.